2012 Annual Report
1a.Objectives (from AD-416):
Objective: Evaluate leukocyte patterns, gene expression profiles, and inflammatory mediators in adipose tissue under influence of dietary manipulation that leads to obesity.
1b.Approach (from AD-416):
The research approach to accomplish this objective employs an established animal model of diet-induced obesity, the C57BL/6J mouse strain, fed diets rich in milk fat or corn oil, compared with low-fat control diets matched for all nutrients except the level of milk fat or corn oil. At specified times of feeding, animals will be analyzed for metabolic changes induced by the diets (e.g., insulin sensitivity, RER), systemic inflammatory changes, and local inflammatory changes in intra-abdominal and subcutaneous adipose tissue depots. The inflammatory changes analyzed include gene (qPCR and/or expression arrays) and protein (ELISA and/or flow cytometry) expression of cytokines, chemokines and adhesion molecules, and leukocyte subsets phenotypically characterized (flow cytometry); and mechanistic studies will be carried out using targeted deletions of proteins potentially key to inflammatory cascades (e.g., CD11c knockouts, gamma delta T cell knockouts, TLR4 knockouts, etc., backcrossed to the C67BL/6J strain). The experimental approach analyzing anti-inflammatory factors (e.g, candidate genes such as IL-1ra or expression array analysis) influenced by changes in diet will utilize this animal model with focus on the early times following initiation of the high fat feeding and on times following reversal of the high-fat diet to a low-fat diet, a time we have found involves rapid resolution of inflammation that precede reductions in fat mass.
The studies this year have focused on the following specific areas of investigation: .
1)The early response of fat tissue in the abdomen to initiating a high milk fat diet. .
2)The importance of some specific genes for the inflammatory response in abdominal fat tissue when mice are maintained on a high milk fat diet. .
3)The importance of lymphocytes and macrophages to the inflammatory response to a high fat diet. Data collected from these studies is discussed.
We have shown that the fat tissue adjacent to the intestinal wall changes within 3 days when male mice are fed a diet containing 45% of the calories as milk fat. The changes include increased production of some genes that are known to promote inflammation and increased numbers of white blood cells called macrophages. An important finding of our current work is that some of these changes arise within the fat tissue, and some arise within the abdominal cavity (called peritoneal cavity) that is outside of the fat tissue. This is the first observation that cells within this cavity respond so soon to a high fat diet. The cells within this cavity that change are macrophages, so we began a study of the response of these cells to components of milk fat. To do this work, we put macrophages in tissue culture and exposed them to two different types of fat. Palmitic acid is a fatty acid that may activate inflammation, and oleic acid is a fatty acid that may have anti-inflammatory activity. Both are found in milk fat. Our results showed that palmitic acid activated some proinflammatory genes in the macrophages and oleic acid activated some anti-inflammatory genes in the macrophages. We are in the process of analyzing the mechanisms by which these fatty acids activate the cells, and we are studying the effects of mixed ratios of these fatty acids on macrophage activation.
We have acquired several strains of mice with specific engineered deficiencies in genes that may influence the development of inflammation, and we are analyzing the effects of these deficiencies on the response of mice to the high milk fat diet. In previous years we have studied the (Toll-like receptor) TLR2 and TLR4 deficient mice. This year we studied mice deficient in MMP12, a very important enzyme produced by macrophages. The mice appear to breed normally, and their development under carefully controlled laboratory conditions appears to be normal when the mice are maintained on normal high protein, low fat diet. Breeding was carried out so that we could compare the MMP12 deficient mice with normal mice from the same litter. These studies are in progress, and preliminary data reveal important differences in glucose tolerance and insulin levels in the MMP12 deficient mice in response to the high fat diet. In addition to the MMP12 deficient mice, we have been analyzing mice that are deficient in a protein called interferon gamma that is important in the inflammatory process. This protein is known to be proinflammatory in many situations, and has been shown to be proinflammatory when mice are fed a long-term high fat diet. Our current data indicate that in contrast to long-term high fat feeding, interferon gamma has an anti-inflammatory role within the first 5 weeks of high fat feeding. This is of particular interest in light of our interest in the yo T cells in adipose tissue. We have shown that these cells produce interferon gamma. So, both the MMP12 and interferon gamma studies are partially completed, and will continue into the next year of this project.
We have been studying three types of lymphocytes to assess their contributions to the inflammation induced by a high fat diet. They are alpha Beta T cells, yo T cells, and NK cells. All three are normally found in fat tissue, and their numbers change when animals are fed a high fat diet. This year, we have studied mice that are deficient in the alpha Beta T cells and found that they show very marked reduction in inflammation when fed a long-term high fat diet, and they do not develop the prediabetic and diabetic changes seen in normal mice. This is very strong evidence that these cells are involved in the pathology induced by obesity. We have found that mice deficient in yo T cells also show some reductions in inflammation when fed the high fat diet, but they appear not to be as important as the alpha Beta T cells. We have begun studies in tissue culture to see if yo T cells will directly alter the functions of fat cells, and have developed the techniques needed for these studies to continue into the next year of this project. The third type of cell, the NK cell, has not been studied in fat tissue, though it is known that there are relatively large numbers of these cells in fat tissue. We have been analyzing the possible functions of these cells in inflammation, and have shown that in some tissues the NK cells serve to control the inflammatory process. We are beginning to analyze the NK cells in fat tissue to see if they serve this function here. These studies will continue into next year, investigating the effects of depleting NK cells on inflammation and prediabetic changes with diet-induced obesity.
The role of alpha Beta T cells in diet induced inflammation. The alpha Beta T cells, an important class of white blood cells that are known to help protect the body from infection, have been found to increase in fat tissue during obesity, but their importance there has been unknown. Researchers at the Children's Nutrition Research Center in Houston, Texas, studied mice that are deficient in these cells by placing them on a high fat diet for 3 months and analyzing tissue inflammation and diabetic changes that occur as the mice become obese. They found that inflammation and diabetic changes were markedly reduced in these mice. These observations provide strong evidence that these cells in normal mice may be stimulated by the high fat diet and instead of protecting the body, actually cause tissue injury.